Method for identification of protease activity inhibitors and assaying the presence of protease activity

ABSTRACT

A system for the identification of proteases and protease inhibitors is provided. The system has at least two components. The first component is a reporter construct with at least one binding site, a transcriptional promoter, an inducible promoter region, and at least one reporter gene, all functionally connected for expression of the reporter gene(s) in functional coordination with a transcriptional activation agent. The second component is a transcriptional activation agent comprising a nucleic acid binding domain, at least one protease substrate domain, and at least one transcriptional activation domain for an inducible promoter. The system allows detection and evaluation of agents affecting protease activity directed to the protease substrate domain. The system also allows for the detection of the presence of proteases in environmental samples.

CROSSREFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/267,386, entitled “BOTULINUM NEUROTOXININHIBITOR IDENTIFICATION METHOD AND SYSTEM” and filed Dec. 7, 2009,which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention generally relates to the field of protease inhibitoridentification assays.

BACKGROUND

Proteases play an important role in biological processes. Proteases,however, can also cause significant harm to biological systemsparticularly those delivered by virus, toxins and pathogenicmicro-organisms. Methods for developing protease inhibitors and assayingfor protease activity particularly in cells is a critical area ofbiotechnology. For example, the Botulinum neurotoxins (BoNTs) are themost potent toxins known (S. S. Arnon, R. Schechter, et al. Jama285:1059-70. (2001); and B. M. Paddle. J Appl Toxicol 23:139-70. (2003).Botulism can be caused by ingestion of food stuff contaminated with thebacteria Clostridium botulinum, colonization of open wounds by thebacterium, or ingestion or respiration of the toxin(s). These toxinsrepresent a serious threat to both military personnel and civilianpopulations (S. C. Clarke. Br J Biomed Sci 62:40-6 (2005); R. P. Hicks,M. G. Hartell, et al. Curr Med Chem 12:667-90 (2005); D. Josko. Clin LabSci 17:30-4 (2004). The lethal dose in humans is <1 ng/kg of bodyweight. J. C. Burnett, E. A. Henchal, et al. Nat Rev Drug Discov4:281-97 (2005); J. C. Burnett, J. J. Schmidt, et al. Bioorg Med Chem13:333-41 (2005); B. M. Paddle J Appl Toxicol 23:139-70 (2003). TheCenters for Disease Control and Prevention has listed these toxins ascategory A (the highest priority) bio-threat agents. Although BoNTs canbe dangerous, they have been recognized as useful medicinal compounds.BoNTs are now established biotherapeutics for a range of physicalailments and cosmetic treatments and are being produced in increasingquantities, both domestically and overseas. R. Bhidayasiri, and D. D.Truong, J. Neurol. Sci. 235:1-9 (2005); C. L. Comellaand and S. L.Pullman. Muscle Nerve 29:628-44 (2004); K. A. Foster. Drug Discov Today10:563-9 (2005); R. G. Glogau. Clin J Pain 18:S191-7 (2002); J. D.Marks. Anesthesiol Clin North America 22:509-32, vii. (2004); C.Montecucco and J. Molgo. Curr Opin Pharmacol 5:274-9 (2005). A negativeconsequence of their usefulness is the increased availability of theneurotoxins for misuse Likewise, increased usage increases thelikelihood of the occurrence of unintended adverse effects duringtreatment. T. R. Cote, A. K. Mohan, et al. J Am Acad Dermatol 53:407-15(2005).

Once inhaled into the lung or ingested into the gastrointestinal tract,the BoNTs are transcytosed across the respiratory epithelium or mucosainto the blood stream, where they can enter the intercellular spaceprior to binding to and entering the peripheral cholinergic presynapticnerve endings. Currently, critical care mechanical ventilation is theonly treatment option once neurons have been affected and diaphragmmuscles cease to function. However, the effects of internalized BoNTscan last for months. R. Eleopra, V. Tugnoli, et al. Neurosci Lett256:135-8 (1998); F. A. Meunier, G. Lisk, et al. Mol Cell Neurosci22:454-66 (2003). As such, long-term mechanical ventilation would beimpractical if even a limited number of individuals were simultaneouslyaffected.

There are seven BoNT serotypes (A-G), which possess different tertiarystructures and significant sequence divergence. Structurally, eachserotype is composed of a 100 KDa heavy chain (HC) and a 50 KDa lightchain (LC). They are synthesized initially as a single polypeptidechain, which is severed by bacterial or host proteases. The chainsremain connected by a disulfide bridge until reaching the reducingcytosolic environment of the neuronal target cells. D. B. Lacy, W. Tepp,et al. Nat Struct Biol 5:898-902 (1998). L. L. Simpson. Annu RevPharmacol Toxicol 44:167-93 (2004). The LC is a zinc-dependentendopeptidase.

Once inhaled into the lung or ingested into the digestive tract, BoNTsare transcytosed across the mucosal epithelium into the blood stream,where they can enter the intracellular space prior to accessingperipheral cholinergic presynaptic nerve endings. The HC serves as adelivery system for the proteolytic LC by binding to neurons andtransporting the LC into the cytosol via the carboxyl terminal half ofthe HC(HC_(C)) and transporting the LC into the cytosol from theendosomes via a pore formed by the aminal terminal half of theHC(HC_(N)). The LC of each BoNT serotype is a protease that cleaves acomponent of the SNARE proteins, which are responsible for acetylcholinecontaining vesicle fusion and release at the neuromuscular junctions. B.R. Singh. Nat. Struct Biol 7:617-9 (2000); and K. J. Turton, A.Chaddock, and K. R. Acharya, Trends Biochem. Sci. 27:552-8 (2002). BoNTserotypes A and E cleave SNAP-25 (synaptosomal-associated protein (25kDa). T. Binz, J. Blasi, et al. J Biol Chem 269:1617-20 (1994).Serotypes B, D, F and G cleave VAMP (vesicle-associated membraneprotein, also referred to as synaptobrevin and VAMP-2). G. Schiavo, F.Benfenati, et al. Nature 359:832-5 (1992); G. Schiavo, C. Malizio, etal. J. Biol. Chem. 269:20213-6 (1994); G. Schiavo, O. Rossetto, et al. JBiol Chem 268:23784-7 (1993); G. Schiavo, C. C. Shone, et al. J BiolChem 268:11516-9 (1993); J. J. Schmidt, and R. G. Stafford. Biochemistry44:4067-73 (2005). Serotype C cleaves both SNAP-25 and syntaxin1a. J.Blasi, E. R. Chapman, et al. Embo J 12:4821-8 (1993). BoNT mediatedcleavage of the SNARE proteins results in flaccid paralysis, bypreventing motor neurons from releasing acetylcholine at theneuromuscular junctions and interrupting the function of autonomicnerves via the inhibition of acetylcholine release as well. Oncediaphragm muscles are affected, breathing is impaired and ultimatelysuffocation results.

The seven BoNT serotypes differ significantly in amino acid sequence.However, the different serotypes adopt similar overall protein folds andaspects of the catalytic core are conserved. M. A. Breidenbachand A. T.Brunger. Trends Mol Med 11:377-81 (2005). The X-ray crystal structuresof BoNT/A and BoNT/B indicate that the areas within 8 Å of thezinc-binding site of these two serotypes are highly homologous with 17of the 22 residues being identical. S. Swaminathan & S. Eswaramoorthy,Nature Structural Biology 7:693-699 (2000). However, significantvariation is observed within 15 Å, including at the zinc-binding pocket,which is buried much more deeply in BoNT/A than in BoNT/B. Therefore,the active sites differ sufficiently among the serotypes, such thatbroad-spectrum potential inhibitors are unlikely. Furthermore, uponbinding, the substrate wraps around the circumference of BoNT LC,creating an unusually large substrate enzyme interface. M. A.Breidenbachand A. T. Brunger. Nature 432:925-9 (2004). BoNT substratespecificity is also determined by its binding of the substrate over thelong substrate/LC protease interface through sites distal to the activesite, which is called “exosite” binding. M. A. Breidenbachand A. T.Brunger. Trends Mol Med 11:377-81 (2005).

Vaccine approaches will likely play a role in biodefense against BoNT.M. P. Byrne and L. A. Smith. Biochimie 82:955-66 (2000). J. B. Park andL. L. Simpson. Expert Rev Vaccines 3:477-87 (2004). However,identification and inoculation of all members of large at riskpopulations prior to exposure is problematic. The development oftherapeutic approaches that are effective post-exposure treatment isessential. Low molecular weight, non-peptidic inhibitors offer the bestopportunity for the development of post-exposure therapeutics.Interruption of later steps in the pathway, and particularly proteolyticsteps, is desirable for post-exposure therapy. Such compounds would haveto be capable of penetrating into the cytoplasm of the intoxicatedneurons and would need to act with specificity.

BRIEF DESCRIPTION OF THE DRAWINGS

The numerous advantages of the present invention may be betterunderstood by those skilled in the art by reference to the accompanyingdrawings in which:

FIGS. 1A and 1B are schematic diagrams of three constructs made inaccordance with one embodiment of the invention and their interactionwith other molecules for assessing the change in the transcriptionsignal of a reporter in the presence of a protease. One constructprovides a Transcriptional Activator agent (“TA”). The TA agentcomprises a Binding Domain (“BD”), a Protease Substrate (“PS”) domain,and a transcriptional Activation Domain (“AD”). The second construct isa Protease Construct (“PC”). The PC comprises a promoter, a regulatorsequence, e.g. TetO, and the sequence of a protease, which proteolyticactivity cleaves the PC of the TA. The third construct is a ReporterConstruct (“RC”). The RC of one preferred embodiment comprises atranscriptional promoter region and the reporter gene(s).

The transcriptional promoter region comprises at least two elements: atleast one binding site (“BS”) sequence that functionally corresponds tothe BD domain of the TA agent and a minimal promoter region having atleast one TATA box sequence. The system illustrated in this figure iscalled the “cleave off” system because when the protease of the PCcleaves the PS, transcription stops and signal decreases.

FIGS. 2A and 2B are schematic representations of the three constructsgenerally described in FIGS. 1A and 1B, for illustration/exemplarypurposes the domains illustrated as part of the TA agent are: the BDderived from transcriptional factor for the Gal4 operon, the PS iseither VAMP2 (amino acids 25-94) or SNAP25 (amino acids 104-206), andthe AD is the nuclear factor κB (“NFκB/AD”). The elements illustrated aspart of the RC in FIG. 1B are: a promoter consisting of at least one BScorresponding to the Gal4 BD of the TA agent and a minimal adenoviruspromoter region comprising the TATA box (E. D. Lewis, J. L. Manley, Mol.Cell. Biol. 5: 2433-2442 (1985). The PC comprises the CMV promoter witha TetO sequence for regulation of expression and the SBP-CFP-BoNT/LC-Asequence for expression of BoNT/A light chain. Other constructs mayinclude the light chains of any botulinum toxin or a protease thatcleaves the PS on the TA agent.

FIGS. 3A and 3B are a schematic representation of a system in accordancewith one embodiment of the present invention in which the BD and AD ofthe TA agent are attached to the end of the PS. Where the PS islocalized to a membrane or kept outside the nucleus of the cell. Whenthe protease is added to the system, it cleaves the PS releasing theBD-AD pair and enhancing transcription of the Reporter Gene (“RG”). Thissystem is referred to as the “cleave on” system.

FIG. 4 a is a schematic representation of the “cleave on” system wherethe PS is VAMP-2 and FIG. 4 b is a schematic representation of the“cleave on” where the PS is SNAP-25.

FIG. 5 is a schematic representation of a TA agent and a RC, which havean additional element to control any leakage of the minimal promoter.The additional element is at least one copy of a transcriptionregulator, in one preferred embodiment the transcription regulator isthe TetO promoter region (5′-tccctatcagtgatagagatc-3′). Specifically, inthe illustrated embodiment, the construct employs four copies of theTetO promoter sequence.

FIG. 6 a bar graph of the results of experiments showing the ratio ofbioluminescence in the presence and absence of tetracycline for stablyintegrated RCs. The clones in this figure do not contain the TA agentconstruct.

FIG. 7 is a bar graph of the results of experiments showing the ratio ofbioluminescence in the presence and absence of tetracycline for stablereporter in the presence of TA agent.

FIG. 8 shows the results of a microplate cell-based assay of cellscontaining a reporter construct and the indicated BD-VAMP-NFκB TA agentin the presence and absence of tetracycline.

FIG. 9 is a bioluminescence assay in accordance with one preferredembodiment of the present invention showing the effect of the indicatedTA agents on YFP (Venus) and GLuc expression.

FIG. 10 is a bar graph of the results of experiments showing theevaluation of stable BoNT/LC-B indicator cell lines.

FIG. 11 is a bar graph and pictures of bioluminescence results of afunctional test of the TA agent constructs.

FIG. 12 is a bar graph showing validation of the cleave off indicatorsystem.

FIG. 13 is a bar graph showing validation of the cleave on system instable cell lines.

SUMMARY OF THE INVENTION

A system for the identification of proteases and protease inhibitors isprovided. The system has at least two components. The first component isa reporter construct with at least one binding site, a transcriptionalpromoter, an inducible promoter region, and at least one reporter gene,all functionally connected for expression of the reporter gene(s) infunctional coordination with a transcriptional activation agent. Thesecond component is a transcriptional activation agent comprising anucleic acid binding domain, at least one protease substrate domain, andat least one transcriptional activation domain for an induciblepromoter. The system allows detection and evaluation of agents affectingprotease activity directed to the protease substrate domain. The systemmay also include at least one protease or protease candidate thatspecifically cleaves the protease substrate domain of thetranscriptional activation agent.

A second preferred embodiment of the present invention is a method toidentify protease inhibitors utilizing the system described above. Yetanother embodiment of the present invention provides for a method toidentify the presence of proteases in an environmental sample utilizingthe system described above.

DETAILED DESCRIPTION

The invention summarized above may be better understood by referring tothe following description, which should be read in conjunction with theaccompanying claims and drawings. The description of embodiments, setout below to enable a person of ordinary skill in the art to make anduse the invention, is not intended to limit the invention, but to serveas particular examples thereof. Those skilled in the art wouldappreciate that they may readily use the concept and specificembodiments disclosed as a basis for modifying or designing alternative,elements, methods and systems for carrying out the present invention.

One embodiment of the present invention provides a novel, cell-basedsystem for identification of protease inhibitors and evaluation ofprotease activity. The components of the system comprise multipleconstructs. As shown FIGS. 1 through 5, three constructs form part ofthe system: a Transcriptional Activation agent (“TA”, sometimes hereinalso referred to as “transactivator” construct), a Reporter Construct(“RC”), and a Protease Construct (“PC”). The three constructs can beutilized in two types of protease evaluation systems. In a “cleave off”system as shown in FIGS. 1 and 2, the product of the PC inactivates theTA, resulting in a decrease in transcription of the product of the RC.In a “cleave on” system as shown in FIGS. 3 and 4, the product of the PCreleases the active portion of the TA agent activating transcription andenhancing signal from the reporter of the RC.

The TA agent is engineered to express a chimeric protein moleculecomprising three elements: a DNA Binding Domain (“BD”), a ProteaseSubstrate domain (“PS”) comprising the cleavage site for at least oneprotease, and a transcription Activation Domain (“AD”). In one preferredembodiment of the present invention, the TA agent is designed so thatthe BD and the transcriptional activation domain AD are on oppositesides of the PS as described in FIGS. 1 and 2. In other embodiments ofthe present invention, the PS is on one end of the BD-AD elements of theTA agent as shown in FIGS. 3 and 4. Whether the system is a “cleave on”or “cleave of” system depends upon the position of the PS in the TAagent.

The TA agent according to one preferred embodiment utilizes botulinumtoxin substrates, such as SNAP-25 or VAMP-2. The selected domains ofSNAP-25 and VAMP-2 in these constructs are sufficient to allow cleavageactivity. Accordingly, domains sufficient to encompass the proteasesubstrate domain of either protein in respect to the BoNT proteases thatnormally cleave the respective substrate are provided. More preferably,the PS domain provided is sufficiently large to at least encompass alsothe exotoxin PS sites. M. A. Breidenbach and A.T.B. TRENDS in MolecularMedicine 11: 376-381 (2005). For VAMP-2, the PS domain would compriseamino acids 25-94 of VAMP-2. Cornille F, Martin L, et al. J Biol. Chem.272:3459-64 (1997); Sikorra S, Henke T, et al. J Biol. Chem.283:21145-52 (2008). For SNAP-25, that domain would comprise amino acids104-206 of SNAP-25. S. Chen and J. T. Barbieri, Journal of BiologicalChemistry 281: 10906-10911 (2006). In one preferred embodiment, thesequence of the AD constructs are BD-SNAP-25-NFκB or BD-VAMP-NFκB. TheSNAP-25 and VAMP-2 fragments utilized lack their palmitoylated residues,thus preventing localization of the TA agent to the plasma membrane orcellular vesicles respectively.

The PC includes a protease that recognizes a Protease Substrate (“PS”)in the TA agent. The PC may be a vector expressing the protease andcapable of being expressed in the host cell containing the TA and RC asshown in FIGS. 1 through 5. In one embodiment of the present invention,the protease is expressed in a vector as described in Example 3 below.In an alternative embodiment, the PC can be a protease or a proteaselike molecule introduced into the cell expressing the TA and RC. Theprotease of the PC cleaves the PS domain of the TA agent. In accordancewith one embodiment, the AD of the TA agent is brought into proximity ofthe promoter on the RC by the BD, promoting transcription of a reporterlocated transcriptionally downstream from the BS of the RC as shown onFIGS. 1 and 2. When the PC is activated, or present in the host system,the proteolytic activity of the protease acts to deactivate and renderineffective the TA agent as a transcriptional enhancer by separating theBD from the AD, as shown in FIGS. 1B and 2B. In a preferred embodiment,the protease is selected from among BoNT A, C and E, and the PS isSNAP-25. In an alternative preferred embodiment, the protease isselected from among BoNT B, D, F and G, and the PS is VAMP-2. In a yetanother preferred embodiment, the BoNT is serotype C and the PS issyntaxin1a (GenBank: AAK54507.2). In one embodiment, the TA may includea domain of syntaxin1a that lacks its c-terminal transmembrane domain(BD-syntaxin1a (1 to 265)-AD). The protease substrate may be any knownprotease substrate. It is expected that various proteases may also beutilized. Examples include the anthrax protease, caspases, alpha virusNSP2 protease, HIV processing proteases, Sumo processing proteases,Ubiquitin processing proteases, ISG15 processing protease, autophagyrelated ATG4 like processing proteases, and Hepatitis C processingproteases.

In accordance to other embodiments, cleavage of the PS domain results inenhanced expression of the reporter gene (the “cleave on” effect) asshown in FIGS. 3B and 4B. If the cleavage releases a unit comprisingboth the BD and AD elements functionally connected, transcription isenhanced. As shown in FIGS. 3B and 4B, the TA agent consisting of a BDand an AD can be kept outside the nucleus by palmitoylated residues onthe protease substrate (PS) domain. In yet further embodiments, theBD-AD pair may be attached to other molecules that keep the BD-ADconstruct outside the nucleus of the cell until the protease from the PCreleases the BD-AD construct, which is transported into the nucleus andthen enhances transcription of the reporter gene. In such arrangementthe protease substrate domain may be attached to the plasma membrane orother vesicular membranes in the cell. The cleavage site of the proteaseis located between the TA consisting of the BD-AD and the extra-nuclearanchoring site of the PS. Thus, when the PS is cleaved by the protease,the BD-AD is freed to enter the nucleus and enhance transcription of theindicator, signaling the presence of the protease. In one embodiment ofthe present invention, expression of the protease in the PC isregulated. For example, a TetO control element may be included upstreamof the protease gene preventing expression of the protease unless theappropriate conditions are present. In one preferred embodiment, theTetO operator is utilized, which prevents expression of the protease inthe absence of Tetracycline. It is contemplated that other controlmechanisms known to individuals of ordinary skill in the art would alsobe appropriate for controlling the expression of the protease in thehost cells.

The RC is a nucleic acid based construct. Preferably, the TA agentand/or the PC are also nucleic acid based constructs that express thetrans-activator molecule and the protease, respectively. However, aperson having ordinary skill in the art would recognize that the TAand/or the PCs may be provided as pre-made proteins to a functionalmammalian cell. Likewise, an artisan skilled in the art can understandthe application of the three construct system in other backgrounds, e.g.a cell-free system, where either or both the TA agent and the PC areprovided as nucleic acid or proteins, where of the three constructs maybe fixed on membranes and so on. In the description, below, the focus ison the preferred embodiment, where each of the constructs is atransgenic genetic construct introduced into a mammalian cell,preferably a human cell.

The RC has one or more BS recognized by the BD of the TA agent, apromoter sequence preferably comprising a TATA box and at least onereporter gene as shown in FIGS. 1 through 4. The BD element of the TAagent binds to the one or more BS elements. In one embodiment of thepresent invention the Gal4 BD is used in the TA agent and thecorresponding Gal4 BS is used in the RC. In another preferred embodimentthe LexA BD and corresponding BS sequence are utilized. Similarly, otheractivation domains from transactivators may be utilized such as B42acidic blob domain, VP16 acidic activity, and p53 acidic activationdomain. J Estojak, R. Brent, E. A. Golemis Molecular and CellularBiology 15:5820-5829 (1995); and H. Lee, K Hun Mok et al. JBC 275:29426-29423 (2000). In one preferred embodiment, the IPR has five copiesof the Gal4 cognate DNA binding sequence located in amino acids 1 to148. It is contemplated that multiple copies of other binding domainrecognition sequences may be utilized. For example, the binding domainsequences (BD) for LexA. The binding sites are usually located 10 to 500bp upstream of the TATA box.

In a preferred embodiment, the BS and promoter sequence constitute anInducible Promoter Region (“IPR”) that is essentially a bipartiteconstruct with a first component being the minimal promoter TATA box,which functions minimally alone and upstream from the minimal promoter,and a second component being at least one BS that significantlyincreases transcription from the bipartite promoter in the presence ofan intact TA agent bound to the BS. In a preferred embodiment, the IPRhas a minimal adenovirus promoter region (E. D. Lewis, J. L. Manley, MolCell Biol 5: 2433-2442 (1985). Utilizing several copies of the BSrecognized by the BD of the TA agent allows for stronger binding of theTA agent to the RC. The number of BS to be provided ranges from 1 toabout 8, preferably about 5. In accordance to the above, preferred BDelement, the corresponding BS is the DNA sequence recognized by the BD.K. H. Young, Biol. Reprod. 58: 302-311 (1998). In this configuration theminimal TATA box promoter region will be able to promote only veryminimal transcription in the absence of binding to the BD region by anadditional transcriptional activator, in this case provided by the BD-ADchimeric protein.

In some instances the first element of the bipartite transcriptionalcontrol region consisting of the minimal promoters such as the TATA boxmay lead to an undesirably high level of transcriptional activity in theabsence of binding of the transcriptional activator containing the BD-ADto the BS sequence. To allow a greater level of control throughsuppressing transcription from the minimal promoter TATA box in theabsence of binding to the BS by a transcriptional activator, anadditional tetracycline regulated repressor or preferably a tetracyclineregulated suppressor element is placed downstream of the minimalpromoter as shown in FIG. 5. This DNA sequence element termed a TetOwill bind a tetracycline repressor protein or a tetracycline suppressorprotein in the absence of tetracycline as shown in FIG. 5. In thepresence of tetracycline the tetracycline responsive repressor orsuppressor protein will be released from the TetO element and relievethe repression of transcription from the bipartite transcriptionalcontrol region containing the BS and minimal TATA region. It iscontemplated that other control elements may be used.

In one exemplary embodiment, a transcriptional control region is locateddownstream of the BS and the promoter region (which promoter region maycomprise a TATA box). In accordance to a preferred embodiment, theelement downstream of the promoter region on the Reporter Construct isat least one copy of a 21-nucleotide TetO promoter region. N. F. J. vanPoppel, J. Welagen, et al. International Journal for Parasitology 36:443-452 (2006). Preferably, the RC comprises at least one to about sixTetO promoter repeats, more preferably about four TetO promoter repeats.When the RC is located in a TetS cell which comprises a tTS geneproduct, transcription over the TetO promoter region is blocked. Apreferred such TetS/tTS cell line is a HeLa cell line derivative, forexample the cell line from Clontech: HEK 293 tTS, Catalog #631146; orHeLa 293 tTS, Catalog #631147. Upon addition of tetracycline, the TetOpromoter is not bound by tTS. In one preferred embodiment of theinvention, the Reporter Construct includes additional components toenhance the efficiency of the method of evaluating protease activity.One such component consists of a transcription silencing or inhibitionsequence that is used to prevent transcription of the reporter productunless the appropriate conditions are present. For example, as shown inFIG. 5, several copies of the Tet operons (TetO) may be placeddown-stream from the promoter. N. F. J. van Poppel, J. Welagen, et al.International Journal for Parasitology. 36:443-452 (2006). If the RC isintroduced into cell lines that express transcription silencer tTS thetranscription of the reporter will be repressed. Addition oftetracycline will remove the tTS from binding to the TetO and thepromoter will be highly activated. A person of ordinary skill in the artwould recognize that other similar transcription inhibitors may beutilized. It is understood that an increase in the number of copies ofthe TetO is directly related to the level of transcription of thereporter, as more copies of the inhibitor bind to the region tighter.

The AD element of the TA agent (in accordance to the preferredembodiment described above, the AD is NFκB) is then free to facilitatetranscription. This additional control level allows for a tightlycontrolled system. For example, absent tetracycline, there is noreporter gene product and the expression is not particularly “leaky.”Background transcriptional levels in the absence of expression the TA orrelease of the BD-NFκB chimera can be measured.

The IPR comprising the above elements is upstream and controlstranscription of one or more reporter genes. In a preferred embodimentof the present invention more than one reporter may be utilized toevaluate protease activity. For example, two different fluorescentmolecule sequences may be included. Other reporter couples may also beutilized, such as a fluorescent reporter and an antibiotic resistancesequence. The two sequences may be translated as separate molecules ormight produce a chimeric product. In one preferred embodiment, the tworeporters are part of a single translation product. In a yet morepreferred embodiment, the two reporter molecules are separated by acleavable linker. In one example, as shown in FIGS. 2 and 4, a Venusgene product is fused to the Gaussia luciferase gene (GLuc) gene productand the two reporter proteins are linked by a “self-cleavage” peptide 2Asequence of the foot-and-mouth disease virus (FMDV). M. D. Ryan and J.Drew, Foot-and-mouth disease virus 2A oligopeptide mediated cleavage ofan artificial polyprotein, The EMBO Journal 13:928-933 (1994). A personof ordinary skill in the art would recognize that other self-cleavagepeptides may be utilized to link the two reporters or that the tworeporters may be active as part of a fused protein product, notnecessitating separation into two protein products. The 2A cleavage siteallows the production of secreted GLuc activity into the medium and cellfluorescence from Venus expression. Inclusion of both reporter genespermits instantaneous examination of cells microscopically for Venus YFPproduction as well as detection of bioluminescence in plate readers.Because the GLuc product is released into the media in which the cellsare grown, over-expression of the GLuc reporter can be easily measuredby methods recognized by a person of ordinary skill in the art. Analternative method to express two proteins from one transcript (one RNAexpressed from one promoter) is to insert the Internal Ribosome EntranceSite (IRES) in between two genes. Yury A. Bochkov and Ann C. PalmenbergBioTechniques 41:283-292 (2006).

The system may be utilized to evaluate the activity of the protease thatspecifically recognizes the PS of the TA agent, in vivo. For example,when the construct is expressed in cells that contain a RC, the level ofexpression of the reporter product indicates the presence of thechimeric BD-AD product, which is a function of the activity of theprotease in the same cell.

When the protease substrate contains trans-membrane components, theeffect of the BD-AD components are disabled. For example, the botulinumneurotoxin protease substrates in their natural form containpalmitoylated residues that localize the proteins to vesicularmembranes. Lane, S. R. and Y. C. Liu. Journal of Neurochemistry 69:1864-1869 (1997). As a result, the PS utilized in the BD-PS-ADconstructs described above exclude the palmitoylated residues of thesubstrate. Localization to the cell membrane can be avoided simply bydeleting palmitoylated residues from the construct. A person of ordinaryskill in the art would recognize that in some embodiments, instead ofexcluding the palmitoylated residues from the construct, the constructmay be engineered to prevent palmitoylation of those residues andinhibit localization of the construct to vesicular membranes.

Palmitoylation and the resulting localization to the cell membrane,however, can also be used in an alternative preferred embodiment of thepresent invention. In such embodiment, a palmitoylated proteasesubstrate is attached to the transcription enhancer domain as shown inFIGS. 3 and 4. This configuration is described below as the BD-AD-PS oras AD-BD-PS where the order of BD-AD and AD-BD are interchangeable.Alternatively, the protease substrate may be attached to thetranscription enhancer element resulting in a PS-BD-AD configuration. Inexamples of these preferred embodiments, the botulinum neurotoxinsubstrate is provided as shown on FIG. 4 a (BD-NFκB-VAMP) and FIG. 4 b(SNAP-25-BD-NFκB), where the BD in this preferred embodiment is the Gal4binding domain. In another preferred embodiment, the full lengthsyntaxin1 a with the BD-AD domains fused to syntaxin1a N-terminus. TheC-terminal transmembrane domain of syntaxin1a anchors theBD-AD-syntaxin1a full length molecule to the membrane of the presynapticterminal.

Due to the potential limitations of the Cleavage-on BoNT/A cleavageassay, one potential solution which represents a separate embodiment ofthis invention, the BD-AD domain may be fused to the protease substratePS in this case SNAP25 amino acids 104 to 206 (lacking the palmitoylatedcysteine residues present in SNAP25, amino acids 95 to 103) which isfurther fused to either syntaxin1a full length molecule to anchor theentire fusion molecule BD-AD-SNAP25 (104 to 206)-syntaxin1a full length(1-288). This arrangement will not only address potential limitations ofthe SNAP25 full length (1-206)-BD-AD Cleavage-on system for BoNT/A butthe BD-AD-SNAP25 (104 to 206)-syntaxin1a full length (1-288) will alsofunction as a Cleavage-on indicator for BoNT/C1 due to cleavage of boththe SNAP-25 and the syntaxin1a molecules and for BoNT/E in SNAP25. Thereare potential advantages to using syntaxin1a to anchor the BD-AD-SNAP25molecule to the presynaptic membrane. The principle advantage is thatthe syntaxin1a targeting and localization to the presynaptic membraneessentially identical to that of SNAP25 provide correct localization ofthe SNAP25 substrate. Additionally the BoNT/A LC is trafficked to thepresynaptic membrane similar to the syntaxin1a trafficking allowinglocalization of protease substrate and BD-AD-SNAP25 (104 to206)-syntaxin1a full length (1-328). In another embodiment of thepresent invention, the TA agent is a BD-AD-SNAP25 (104-206)-VAMP-2construct. The BD-AD-SNAP25 (104-206)-VAMP-2 construct is a universalbotulinum protease system that can be utilized as an assay foressentially all BoNT serotypes (BoNT/A, C1, and E cleave SNAP-25 andBoNT/B, D, F, and G cleave VAMP-2).

The reporter sequence of the RC may correspond to the sequence afluorescent protein, a bioluminescent protein or any other protein thatallows for the quantification of a signal upon expression of the gene.It is contemplated that yellow fluorescent protein (YFP), greenfluorescent protein (GFP), cyan fluorescent protein (CFP); bluefluorescent protein (BFP), red fluorescent protein (RFP) and fluorescingmutants thereof, may also be utilized. Bioluminescent proteins such asGaussia luciferase, renilla luciferase, click beetle, and fireflyluciferase may also be used to quantify the activity of the reportervector. In one preferred embodiment, the reporter sequence may consistof the Venus yellow fluorescent protein. Nagai T., Ibata K., Park E. S.,et al. Nature Biotechnol 20: 87-90 (2002).

The system may be utilized to create a genetically engineered cell linecontaining one or more of the constructs described above. The constructsmay be incorporated into one or more vectors for expression in aparticular type of cell. The constructs may be stably integrated in thecell, or may reside on transformation vectors. The methods and vectorsare well known in the art. The methodologies used for transfection andtransduction into cells are well known in the art. Laura Bonetta, TheInside Scoop—Evaluating Gene Delivery Methods, Nature Methods 2:875-883(2005). In a preferred embodiment, one or more of the constructs areintegrated via lentiviral vectors. In a further preferred embodiment,the lentiviral vectors are self-inactivated (“SIN”) lentiviral vectors.A person of ordinary skill in the art would recognize that the vectormay include other selection markers such as antibiotic resistancemarkers in order to distinguish cells that contain the constructs fromthose that do not.

Another preferred embodiment of the present invention provides a methodfor creating a genetically engineered cell line. In a first step of themethod, eukaryotic cells, such as 293-tTS cells, are transduced with avector containing the RC comprising a regulated reporter gene, expressedfrom a minimal promoter controlled by five copies of the Gal4 BS. INother preferred embodiments, four copies of the synthetic tetracyclineoperator are also included (“the G5TO4 promoter”) as described above.

The system is used to evaluate the activity of specific proteases, suchas botulinum neurotoxins. In the first step, a lentivirus vectorcontaining the RC with the Gal5/TO4 promoter and the Venus and GLucgenes is transfected into mammalian 293-Ts cells. The cells are thentransfected with a lentivirus vector containing either the BD-SNAP-25-ADconstruct or the BD-VAMP-2-AD construct. The construct is stablyintegrated. The cell line is engineered to further comprise a geneconstruct encoding BoNT/LC-B to generate the final reporter cell linefor evaluating the activity of the various botulinum neurotoxinproteases. In these final cell strains, expression of BoNT/LC cleavesthe SNAP-25 or VAMP-based transactivator fusion protein, separating theDNA binding domain from the activator domain and, consequently, cellsfail to express the Venus and luciferase reporter genes. Alternatively,the protease is transduced into the cell. The same method may beutilized for identifying the activity of other protease-substrate orbinding domain-binding site couples, as described above.

The reporter cell lines containing the RC, TA agent, and PC, areutilized to identify protease inhibitors. In one preferred embodiment,the cell lines are utilized for high throughput screening of proteaseinhibitors, such as botulinum neurotoxin inhibitors. When intact, thechimeric transcription factor activates the G5 or G5TO4 promoterresulting in expression of the Venus and GLuc reporter genes, and whencleaved by the botulinum neurotoxin light chain, the expression of thereporter genes is turned off. As described previously, this system isreferred to as a “cleave-off” system and is ideal for small moleculeBoNT/LC inhibitor screening because inhibition of BoNT will result in anincrease in reporter signal (“gain-of-signal” assay), reducing thefrequency at which false positives are detected. In the presence ofBoNT/LC inhibitors, the transcription factor will no longer be cleaved,resulting in restoration of the expression of the Venus and GLucindicators.

The cell lines are used in a high throughput screening assay, where thesystem is exposed to potential inhibitors. In one embodiment of thepresent invention, the systems may be utilized to identify inhibitorspresent in available chemical libraries or by testing specific moleculesof interest. One such method utilizing libraries is discussed inExamples 4 through 6.

One embodiment of the present invention presents a cellular,gain-of-signal, bioluminescent, reporter screen. In a preferredembodiment, the present invention identifies endopeptidase inhibitors ofneurotoxins, such as BoNT/A LC and BoNT/B LC, through cell-basedreporter HTS. These endopeptidase inhibitors are small molecules, whichinhibit neurotoxins, such as BoNT/A or BoNT/B. The engineered cell linesused in accordance to one preferred embodiment exhibit a low basalreporter signal, but produce a much higher amplified light signal (>10×)when small molecules inhibit the peptidase activity of the BoNT/LCs.This approach provides a means to identify inhibitors that are active incells against BoNT/LC interacting with SNAP-25, VAMP-2, syntaxin1a, andother neurotoxins. Each cell-based BoNT/LC or HTS screening assayprovides a convenient counter screen for the other assay. Likewise,employing serially a cleave on and a cleave off assays may serve ascounter screening assays. The purpose of these counter screen assays isto determine, for example, the mechanism of action in accordance to theinvention as opposed to other, general toxicity phenomena. Such testingof the system includes cytotoxicity assays or determination of cleavedtranscriptional activator molecules, and quickly remove false positivesand rapidly identify the most selective and non-toxic neurotoxininhibitors. One manner of screening false positives includes theanalysis of the transcriptional activator molecule in a system thatseems to have affected the expression of the reporter molecule. Thescreens for the false positives (e.g. inhibitors that worked by somemechanism unrelated to release or break down of the TA molecule) relayon, for example, the analysis of the size of the TA molecule by aseparation column and antibodies recognizing the TA molecule. Therefore,one embodiment of the invention provides methods for the identificationof “drug-like” small molecules, which inhibit neurotoxin cleavage of itssubstrates, such as SNAP-25, VAMP-2, syntaxin1a, in neurons through cellbased HTS.

The cell-based screening approach described here provides significantbenefits over any in vitro enzymatic screens, since compounds must reachthe intracellular milieu and inhibit neurotoxins, such as BoNT/A orBoNT/B, in the cytosol from cleaving their substrates, such as SNAP-25,VAMP-2, syntaxin1a. Therefore, both the toxin and its substrate are in aclinical, in vivo milieu. The toxin function is very likely differentwithin the cell as opposed to cell free enzymatic activity. The use oflarge substrate fragments of 70-100 amino acid residues is one of thekey advantages to these cell based assays. Since the active site mayencompass proteins larger than the exosites, it allows the detection ofcleavage at sites not normally considered an exosite.

The method and system disclosed herein may be used to identify andprioritize inhibitors of various neurotoxins, such as botulinumneurotoxins A and B for optimization into therapeutics. The method maybe further used to construct, validate, and apply mammalian cell-basedprimary reporter screens for inhibitors of neurotoxins, such as BoNT/Aand BoNT/B, to libraries of diverse compounds. Hits may be confirmed byre-assay in triplicate, and false positives may be eliminated by usingmultiple BoNT-based assays or non-toxin assays as counter-screens foreach other and the other methods as described above. The methoddisclosed in this application further provides for a cellular,gain-of-signal, reporter screen. These screens may be applied librariesof compounds and follow-up with biochemical assays as a secondaryvalidation to identify potential inhibitors. The validated hits may becharacterized thoroughly to ensure that they act specifically on thebotulinum neurotoxins both in vitro and in neuronal cell model systemsand that they exhibit minimal cytotoxicity.

In one embodiment the systems and methods described herein provides forthe identification and development of highly potent small moleculeinhibitors for the treatment of BoNT induced poisoning. Small moleculeBoNT LC inhibitors can penetrate neurons and provide protection bothpre- and post-toxin exposure. In other embodiments the systems andmethods described here may be used for identification of small moleculeinhibitors to other protease substrate pairs that may be important incausing pathogenic states. Such protease substrate pairs may includeanthrax lethal factor, caspases, ubiquitin proteases, sumo proteases,ubiquitin-like molecule processing protease, autophagy relatedprocessing proteases such as ATG4, viral encoded proteases such as alphavirus NSP2 and HIV proteases.

The BoNTs are but one example of the many ways in which a system toidentify protease inhibitors may be utilized. A person of ordinary skillin the art would recognize that the constructs and methods describedherein may be utilized for the evaluation of other proteases, theiractivity and their inhibitors. While the range of possibilities mayinclude nearly any substrate and protease combination some specificexample would include anthrax lethal factor zinc metalloprotease (LF)and its cognate substrate MAPKK, viral processing proteases such as theNSP2 protease of alpha viruses and its cognate substrate NSP1-4,ubiquitin and ubiquitin like molecule processing proteases, caspases,ubiquitin proteases, sumo proteases, ubiquitin-like molecule processingprotease, autophagy related processing proteases such as ATG4, viralencoded proteases such as alpha virus NSP2 and HIV proteases. For thescreening of small molecule inhibitors, cell based systems that have asuppressed signal when the protease is active in cells but generate anincrease in signal when the protease is inhibited by small molecules incells is preferable for the rapid high-throughput screen of smallmolecule inhibitors. This preference is due to the fact that an increasein signal in the presence of a “positive hit” or the presence of anactive protease inhibitor compound is more effective in high throughputscreening (HTS) generally.

Means for detecting the presence of a protease activity in cells may beimportant. For instance if a mass exposure to BoNT were to occur, rapidtriage of those requiring immediate therapy with limited resources vs.those who may not have actually been exposed but feel ill (so calledwalking well) may be needed. Similarly the current “gold standard” orprimary means of assaying the potency and efficacy of BoNTpharmaceutical preparations relies on the injection of toxin into micefor establishment of mouse LD50 units. This method requires extensiveuse of live animals in a lethal assay. It would be desirable to limit oreliminate the use of such live animal assays. A cell based assay whichmight reliably detect the presence and quantify the activity of the BoNTis needed. Cell based assays for detecting the presence of a protease inthe cells such as the BoNT LC protease are best configured with a systemthat turns on an indicator signal or signals with the presences of theprotease in the cell. In one embodiment of the present invention, cellsexpressing the TA agent and RC in the “cleave on” configuration(BD-AD-PS) are utilized to identify the presence of known proteases in asample. In such embodiment, an environmental sample is presented tocells containing the TA and RC constructs. If target protease, e.g.,BoNT/LC A, is present, it will enter the cell, cleave the TA at the PS,releasing the AD-BD fragment, which in turn enhance transcription of thereporter gene in the RC.

Example 1 Expression of pBD-NFκB in Mammalian Cells

A RC in accordance with one embodiment of the present invention,containing a synthetic promoter G5/TO4 was transfected by a lentiviralvector into cells. The RC was co-transfected with a plasmid expressing aGal4 binding domain fused to the NFκB activator fusion (pBD-NFκB) (theTA agent) into HeLa-tTS cells. The transfected cells constitutivelyexpress the tetracycline transcription silencer tTS (Clontech). Thecells were grown in a 6-well plate to approximately 80% confluence. Sixhours after transfection, 1 ug/ml of tetracycline was added to the mediaof the test cells. Tetracycline was not added to the media of cells usedas a control. Culture medium was collected two days post-transfectionfor the Gaussia luciferase (GLuc) assay. Monique Verhaegen and TheodoreK. Christopoulos Anal. Chem., 74:4378-4385 (2002). Venus expression wasobserved with a fluorescent microscope. The relative light units (RLU)of the cells cultured in medium containing 1 μg/ml tetracycline is 16fold higher than that of cells cultured in the absence of tetracyclineas measured by luminometer. Additionally, there is Venus expression inthe cells cultured in medium containing tetracycline but no Venusexpression in cells grown in medium lacking tetracycline. Thus, (1) thesynthetic promoter G5TO4 is functional. It can be highly activated by atransactivator, such as BD-NFκB that binds to Gal4 binding sites. BothGLuc and YFP expression are highly activated in the presence oftetracycline; (2) The GLuc gene is a very sensitive and convenientreporter. In these experiments, ninety-five percent of Gaussialuciferase is secreted into the culture medium; thus, GLuc activity canbe directly measured from culture medium by a luminometer. In light ofthis controlled reporter gene expression, low and measurable backgroundsof reporter gene expression, and convenient use, the promoter-reportersystem described in this application is useful for high throughputscreening.

The novel RC was transduced into 293-tTS cells by lentiviral vector.Lentiviral vector particles carrying the RC were produced byco-transfecting 3.5 μg of the transducing plasmid with 7.1 μg HIV-1gag-pol helper construct (Synaptic Research), and 2.8 μg VSV-Gexpression plasmid (Synaptic Research) onto 80-90% confluent 293FT cells(Invitrogen) cultured in 100 mm plates. Culture medium that containedthe budded viral vectors was collected 48 hours after transfection andwas cleared of cell debris by centrifugation at 2,000 RPM for 10 minutesat 4° C. (Sorvall RT 600D). The cleared viral supernatant was furtherconcentrated by ultracentrifugation at 25,000 RPM for 90 minutes at 4°C. (Beckman Coulter Optima™ XL-100K). Lastly, the viral vector pelletwas soaked in 50 μl ( 1/200 the original volume) of culture mediumovernight, resuspended, and stored at −85° C. until needed fortransduction. The resulting viral vector particles were used totransduce 293T-tTS cells (Clontech®) that constitutively express thetetracycline transcription silencer tTS. Single cell colonies werecloned by cloning rings and tested for functionality by transienttransfection of the expanded cells with a pBD-NFκB construct, whichexpresses the chimeric transactivator BD-NFκB. The transfected cellswere cultured in the presence and absence of 1 μg/ml tetracycline. Twodays after tetracycline induction, YFP fluorescence (Venus geneexpression) was observed with a fluorescent microscope and Gaussialuciferase gene expression was measured by a luminometer.

We analyzed 25 clones, and results of six clones are shown in FIG. 6. Weselected clone #17, which exhibits very low basal activity and can beactivated more than 14 fold by the addition of tetracycline.Accordingly, the reporter molecules were induced by the activator agentand the system has the necessary attributes for high throughputscreening

Example 2 tTS Cells Comprising the RC and TA Agent

A gene for the regulatory component, a transactivator chimeric fusionprotein consisting of the appropriate BoNT substrate, SNAP-25 for BoNT/Aand VAMP-2 for BoNT/B, sandwiched between a Gal4 DNA binding domain(amino acids 1-148) (Gal4/BD or BD) and the NFκB transactivation domain(NFκB/AD or AD) was transduced into the cells that have the novelReporter construct as described in Example 1.

Example 2A

The BD-PS-AD constructs, in which the protein substrate does not containpalmitoylated residues, were constructed synthetically and introduced incells containing the RC. The TA agent encoding either 103 amino acidresidues around the cleavage site of SNAP-25 (residues 104-206) or 70amino acid residues around the cleavage site of VAMP-2 (residues 25-94)fused between the Gal4 DNA binding domain and the NFκB transactivatordomain were used. The reporter cell line clone #17 from Example 1 wasfurther transduced with a lentiviral vector that carries theBD-VAMP-NFκB transactivator gene construct. Six single cell clones wereselected and analyzed for the ratio of bioluminescence in the presenceand absence of tetracycline. See FIG. 7. The transduced cells weresubjected to appropriate selection (G418, blasticidin, and puromycin),and single-cell clones carrying stable integrations of both the reporterand the VAMP-2 transactivator fusion were obtained. The reporter gene inclone #32 was routinely/repeatedly activated more than 200-fold when 1μg/ml tetracycline was added to the culture medium.

A similar process is used to create a cell lines containing otherchimeric transactivator constructs. For example, the cell linescontaining the reporter vector are further transfected with aBD-SNAP25-NFκB or a BD-syntaxin1a-NFκB gene construct. Alternatively,more than one transcriptional activator construct, each containinganother BoNT substrate, is created. In addition, the binding domain (BD)and the transactivation domain (NFκB) may be replaced with any DNAbinding domain and transactivation domain as long as the binding sitesof the Reporter construct are changed correspondingly. Single cellclones carrying the RC and TA agent are selected and their functionalityare evaluated as described for clone #32.

The functionality of clone #32 was analyzed in a 96-well microplate inorder to demonstrate that signal strength and the signal:backgroundratio are adequate for use in high throughput screening. Duplicates ofthree groups of 3 wells each were seeded with a low (group 1), medium(group 2), and high (group 3) density of cells. One day after culturingcells in the presence or absence of 1 μg/ml tetracycline, 5 μl of 5-folddiluted culture medium was transferred to a second microplate andluminescence was measured in a plate reader. The luciferase activity inthe medium of cells treated with tetracycline was 200-fold higher thanthat observed from culture medium of cells not treated withtetracycline. See FIG. 8. That result was consistent for each member ofeach of the three groups. In addition, the basal activity in cellsnon-treated with tetracycline was extremely low. Luminescence wasmeasured. The luminescence was increased 200 fold by transactivation inthe presence of tetracycline and the intact DB-PS-TA construct. See FIG.8.

Example 2B

The “cleave-on” method was evaluated by the transcriptional activatorconstructs encoding either 103 amino acid residues around the cleavagesite of SNAP-25 (residues 104-206) or 70 amino acid residues around thecleavage site of VAMP-2 (residues 25-94) fused upstream or downstream ofthe chimeric transactivator BD-NFκB/AD, respectively, as shown in FIGS.3 and 4. As a result of the palmitoylated residues of the botulinumsubstrates, the SNAP-25 and VAMP-2 segments are expressed on thecellular presynaptic plasma membrane tethering BD-NFκB/AD to themembrane. FIG. 9 shows examples of the results of experiments conductedusing both configurations.

Example 3 Further Validation of Constructs in Stable Cell Lines

Validation of the cleave off system utilizing the VAMP2-based TAconstruct and BoNT/LC-B as the PC, and the TetO containing RC wasaccomplished in stable cell lines. Clone #32, which carries stableintegrations of both the reporter and the VAMP2 transactivator, was usedto construct the final inducible indicator cell line by lentiviraltransduction of the BoNT/LC-B PC. With all three components of theindicator system (RC, TA, and PC) stably incorporated into this completecell line (Clone #12), the system was tested by evaluating the decreasein GLuc expression over the course of 48 hours immediately followinginduction with tetracycline. GLuc assays were performed before replacingthe media with fresh culture media every 24 h. GLuc activity expressedin terms of RLU was measured using a luminometer and showed anapproximate 15-fold reduction in GLuc signal after 48 hours induction ofthe BoNT/B LC from the Tet-regulated PC FIG. 10. This resultdemonstrates that the fully assembled system with the RC, cleave-off TA,and the PC is suitable for high-throughput screening of BoNT/B LCinhibitors. The system in this configuration will exhibit an increase insignal with inhibition of the LC protease.

Validation of the cleave-on system utilizing the VAMP2-based TAconstruct and BoNT/LC-B as the PC, and the TetO containing RC was alsoaccomplished in stable cell lines using the same procedure describedabove. The results show a nearly 40-fold increase in GLuc signal after48 hours. FIG. 10. The system in this configuration will exhibit adecrease in signal with inhibition of the LC protease, but if moreappropriate for detecting the presence of BoNT/LC-B protease.

Example 4 Cleave Off System Reduces Gene Expression

To demonstrate their functionality, the SNAP-25 and VAMP-2 chimerictranscriptional activators were cloned into self-inactivated (SIN)lentiviral vectors and co-transfected into 293T-tTS cells with thereporter construct together with various BoNT/LC gene constructs(wild-type LC-A, an inactive mutant LC-A, and wild-type LC-B). The cellswere treated with 1 μg/ml tetracycline. Gaussia luciferase (GLuc)activity was measured with a luminometer and Venus YFP expression wasobserved by fluorescent microscopy. The results are shown in FIG. 9. Theresults confirmed that the reporter is turned off by cleavage of thetransactivator. The chimeric BD-SNAP25-NFκB transactivator stronglyactivates the G5TO4 promoter when co-transfected with inactive mutantBoNT/A-LC mLC-A and Gal4/BD-SNAP25-NFκB/AD, which was used to mimic thepresence of a potent BoNT/LC-A inhibitor, but not when co-transfectedwith the wild-type BoNT/A-LC LC-A and Gal4/BD-SNAP25-NFκB./AD. Thechimeric Gal4/BD-VAMP-NFκB transactivator/AD transactivates the G5TO4promoter when transfected with the BoNT/A-LC expressing plasmid becauseit is not cleaved by BoNT/A-LC. The chimeric transcription activatorconstruct Gal4/BD-VAMP-NFκB/AD does not activate the reporter whenco-transfected with wild-type BoNT/LC-B LC-B and Gal4/BD-VAMP-NFκB/ADbecause the transcription factor is cleaved. The Gaussia luciferaseactivities expressed as RLU values are consistent with the visualizedYFP fluorescence and provide quantitative measurements of the reporterresponse—20-fold and 23-fold in the SNAP-25 and VAMP transactivatorsystems, respectively, for uncleaved vs. cleaved transactivators.

Two BoNT constructs consist of the streptavidin binding protein (SBP),the cyan fluorescent protein (CFP), and BoNT/A-LC or BoNT/B-LC fusedsequentially and in frame. These two constructs, SBP-CFP-BoNT/A-LC andSBP-CFP-BoNT/B-LC, have been cloned into the lentiviral vectorpLenti4/TO/V5-DEST (Invitrogen, Inc., Catalog No. K4965-00). The fusiongenes are expressed from a modified CMV promoter that has two copies ofTet operator inserted immediately upstream of the TATA box. Binding ofthe Tet responsive repressor (TRex, Invitrogen) or transcriptionsilencer tTS (Clontech) silences the promoter. However, in the presenceof tetracycline the TRex or tTS fails to bind the Tet operator, and theCMV promoter is fully active. The constructs were transfected into293-tTS cells seeded in a 6-well plate. Expression of the BoNT/LC wasdetected by observing CFP expression by fluorescence microscopy. Theselentiviral constructs of BoNT/A-LC and BoNT/B-LC may be used to completethe construction of the reporter cell lines.

Example 5 Complementing Stable Reporter Cell Lines with TA and PC A)VAMP Cleave-Off

In order to evaluate the biological functionality of the reporterconstruction (RC) without TetO, this RC was stably integrated into HEK293 cells with lentiviral vectors as described previously. Using thisstable reporter cell line as a platform, the VAMP cleave-off system wastested by complementation with the corresponding PC (BoNT/LC-B) and TA(BD-VAMP2-AD) plasmids using transient transfection. Immediatelyfollowing transfection, the time-course expression of both Venus (YFP)and GLuc from the reporter construct was assayed. Specifically, thereporter cells were grown in 6-well sterile polylysine coated plates toapproximately 70% confluence in 2 ml of complete growth medium. Fortransfection with transcriptional activator BD-VAMP2(25-94)—NFκB plasmidalone, 4 μg of plasmid DNA was transfected using CalPhos Kit (ClontechLaboratories Inc.). For cells, co-transfected with BD-VAMP2-AD andBoNT/LC-B, a 1:3 ratio of TA:PC plasmid DNA was used. Plates wereincubated at 37° C. overnight in CO₂ incubator. After 12 h, media wasreplaced with 2 ml fresh complete growth media and 5 ug/ml oftetracycline is added to the media to initiate the expression ofBoNT-LC-B. Cells transfected only with BoNT/LC-B were used as positivecontrol for LC-B expression. Tetracycline was not added to the media ofcells transfected with only BD-VAMP2-AD, used as a control. An aliquotof the culture medium was collected at 24 h, 48 h and 72 h for Gaussialuciferase (GLuc) assays before replacing the media with fresh culturemedia every 24 h. GLuc activity expressed in terms of RLU was measuredusing a luminometer and Venus YFP fluorescence was monitored by afluorescent microscope. Cells scraped from the plate surface fromdifferent wells at 24 h, 48 h, 72 h time intervals were washed twicewith PBS and centrifuged at 10,000 rpm for 10 min at 4° C. then lysedwith a gel loading Samples were run on SDS-PAGE gels and thentransferred to PVDF membranes for Western blot analysis with rabbitanti-GFP (Santa Cruz Biotechnology) as primary and AP-conjugatedanti-rabbit IgG as secondary antibody. FIG. 11. The success of the cellsystem with RC lacking the TetO, cleave-off VAMP TA, and BoNT/B LC PC,to demonstrate a decrease in signal with the cleavage of the VAMP TA bythe BoNT/B LC demonstrates that this system is also appropriate forhigh-throughput screening of BoNT/B LC inhibitors.

B) SNAP25 Cleave-Off

Using the same stable reporter cell line described previously, theSNAP25 cleave-off system was tested by complementation with thecorresponding PC and/or TA plasmids using transient transfection. Again,immediately following transfection, the time-course expression of bothVenus (YFP) and GLuc from the reporter construct was assayed.Specifically, reporter cells are grown in 6-well sterile polylysinecoated plates to approximately 70% confluence in 2 ml of complete growthmedium without antibiotics and with serum. For transfection withtranscriptional activator BD-SNAP25(104-206)—NFκB plasmid alone, 4 μg ofplasmid DNA was transfected using Lipofectamine™ 2000 (Invitrogen). Forcells co-transfected with BD-SNAP25-NFκB and BoNT/LcA, 4 μg of eachplasmid DNA is used per well. Six hours after transfection, 5 ug/ml oftetracycline was added to the media to initiate the expression ofBoNT-LC-A. Cells transfected only with BoNT/LC-A were used as positivecontrol for LC-A expression. Tetracycline was not added to the media ofcells transfected with only BD-SNAP25-NFκB, used as a control. Analiquot of the culture medium was collected at 24 h, 48 h and 72 h forGaussia luciferase (GLuc) assays before replacing the media with freshculture media every 24 h. GLuc activity expressed in terms of RLU wasmeasured using a luminometer and Venus fluorescence was be monitored bya fluorescent microscope. Cells scraped from the plate surface fromdifferent wells at 24 h, 48 h, 72 h time intervals were washed twicewith PBS and centrifuged at 10,000 rpm for 10 min at 4° C. then lysedwith a gel loading buffer. Samples were run on SDS-PAGE gels and thentransferred to PVDF membranes for Western blot analysis with rabbitanti-GFP (Santa Cruz Biotechnology) as primary and AP-conjugatedanti-rabbit IgG as secondary antibody. FIG. 11.

C) SNAP25 Cleave-Off—Stable Cell Line with RC (Tetracycline Regulated)

A similar experimental procedure was followed to validate the stablereporter cell line that is regulated by TetO (Clone #17). In this case,Clone #17 was transduced by lentivirus with the BD-SNAP25-AD, which wassubsequently complemented with BoNT/LC-A tet-inducible PC by transienttransfection. Upon exposure to tetracycline, the GLuc signal decreaseddramatically during the first 48 hours. This large decrease in signalwith induction of the BoNT/LC-A LC shows the system will work for thedrug screening approach and assaying decay of BoNT/LC-A.

Example 6 Expression with or without Tetracycline

In transient transfection and stable transduction of the RC withoutTetO, SNAP25 (1-206)-BD-AD as the TA, and BoNT/E LC as the PC, theluciferase signal increases at least 2 fold after cleavage of the SNAP25(1-206)-BD-AD by the BoNT/E LC despite a high basal signal level asshown in FIG. 13. The carboxyl terminal SNAP25 cleavage product afterBoNT/E LC cleavage is stable. Thus there is not degradation of the BD-ADafter cleavage like there is with the BoNT/A cleavage. Due to high baseline signal with the SNAP25 (1-206)-BD-AD and rapid degradation of theC-terminal SNAP25 (1-206)-BD-AD, a different SNAP23 cleave-on system isused. This system consists of BD-AD-SNAP25(104-206)-VAMP full length orBD-AD-SNAP25 (104-206)-syntaxin1a full length.

Example 7 An Alternative Indicator System to Perform SNAP25 Cleave-onAssays

Any TA construct comprised of the truncated form of SNAP25(104-206)lacks palmytolation and, as a result, is not inherently capable ofmembrane localization. The fusion of this TA to a membrane anchor, suchas VAMP2 or syntaxin 1a, allows for an alternative method to performcleave-on studies. Possible TA configurations areBD-AD-SNAP25(104-206)-VAMP2 or BD-AD-SNAP25(104-206)-syntaxin1a.

These fusion constructs are tested in the same manner as before: bytransient transfection of the stable reporter cell line (lacking TetO)with both the fusion TA and either BoNT/LC-A or -B. As with allcleave-on systems, the reporter signal is low at baseline in the absenceof tetracycline, but will increase upon proteolytic cleavage of the TA.After adding tetracycline, an aliquot of the culture medium is collectedafter 24 h, 48 h and 72 h for Gaussia luciferase (GLuc) assays beforereplacing the media with fresh culture media. GLuc activity expressed interms of RLU was measured using a luminometer and Venus fluorescence wasbe monitored by a fluorescent microscope. The major advantage of thesefused TA constructs is the ability for them to acts as universaldetectors of numerous BoNT/LC serotypes. Thus, the potential for asingle indicator cell line to detect the presence of any LC is realized.If the system is created in a cell line that has receptors for the BoNTtoxins and can internalize the toxins efficiently, this cell line canfunction as a high affinity sensitive cell based biodetector for thepresence of fully active BoNT. If a suitable cell line can not be foundthat has adequate ability to take up toxins from the environment tosensitively detect the toxins, then the affinity and sensitivity of thecell line to toxin can be increased by making a stable cell lineoverexpressing the necessary protein receptor and ganglioside componentof the BoNT toxin cellular receptor.

Example 8 Indicator System to Detect Inhibitors of Anthrax Protease

In one example, a TA is constructed such that the PS is the full-lengthmitogen-activate protein kinase kinase (MEK1) (NCBI Reference Sequence:NP_(—)002746) in the form BS-MEK1-AD. A. P. Chopra, S. A. Boone, et al.JBC 278:9402-9406 (2003). This TA construct is similarly transferred tothe stable reporter cell line lacking TetO—used in a Stable Cell Linewith RC (Not Tetracycline Regulated) example—by transient transfection.Accordingly, the Tet-inducible PC in this case is anthrax lethal factor(LF) protease (NCBI Reference Sequence: AAY15237), which cleaves MEK1.With all constructs present in the reporter cell, inhibitors of anthraxLF protease are screened and success candidates are chosen based on anincrease in reporter signals from Venus and GLuc.

Example 9 Indicator System to Detect Inhibitors of Ubiquitin-RelatedProteases

In another example, a TA is constructed such that the PS is the humansmall ubiquitin-related modifier1 (SUMO1) protein with the carboxylterminal 5-AA (NCBI Reference Sequence: ABM87155). The 5-AA C-terminalpeptide is cleaved by the (ubiquitin-like)-specific protease (ULP1),thus a PC construct is constructed with ULP1 as the protease (NCBIReference Sequence: AAG33252). Both the PC and TA construct aresimilarly transferred to the stable reporter cell line lacking TetO bytransient transfection. When the all three components, RC, TA cleave-offSumo construct, and the ULP1 PC are expressed, the luciferase signal isdiminished. This system is appropriate for high-throughput screening ofSUMO protease inhibitors.

Example 10 Screening Protease Inhibitors

The final reporter cell lines is functionally validated by smallmolecule inhibitors of siRNA knock-down. For the BoNT/A-LC screeningassay, established inhibitors (e.g., either hydroxamate compounds) areused. Since there is no small molecule inhibitors known for BoNT/B-LC,siRNAs are used that target BoNT/B-LC from Dahrmacon/Thermo-Fisher. Inone embodiment of the present invention, three siRNAs per target may bedeveloped. In addition, a scrambled siRNA is used as a control, whichensures that the knock-down is real and specific and there in nooff-target effect. 293T cells are co-transfected with the siRNA and theBoNT/A-LC or BoNT/B-LC plasmids and Western blot analysis are used tochoose the most effective siRNA for validating the final reporter celllines. Then both the effective and the scrambled siRNA are used totransfect the final reporter cells. Transfection 1 μg/ml of tetracyclineis added to the culture medium to initiate expression of BoNT/LCimmediately after transfection. An aliquot of the culture medium iscollected at day 1 through day 4 for luciferase assays. Venusfluorescence may be monitored by a fluorescent microscope. Theexpression of the reporters (both luciferase and Venus fluorescence) inthe final reporter cells are restored by the effective siRNA.

The reporter screens are optimized by running microplates with halfpositive (BoNT/A-LC inhibitor or siRNA for BoNT/B-LC) and half negative(DMSO only) controls and measuring the Z′ value. Conditions used todetermine the effectiveness of each reporter strain include the densityof microplate (96-well or 384-well), concentration of compound to betested, DMSO concentration tolerance, temperature, degree of confluenceof reporter strain before addition of test compounds, time of incubationin microplates before reading luminescence, and quantity of mediumwithdrawn for luciferase assay. Conditions may be changed to achieve anoptimal Z′ factor >0.5 J. H. Zhang, T. D. Chung, and K. R. Oldenburg, J.Biomol. Screen 4:67-73 (1999). for each screen. In one preferredembodiment of the present invention the screens are conducted in384-well dishes. 96-well dishes may be used if necessary to maintainadequate Z′ factor values.

Reporter strains are grown and seeded into 96- or 384-well opaque whitescreening plates using a sterile Wellmate Microplate reagent dispenser(ThermoFisher, Inc.). For inhibitor screening, compound master platesare thawed at room temperature on the day of the screen, and apredetermined quantity of compound is added by using a Sciclone ALH 3000liquid handling robot (Caliper, Inc.) and a Twister II MicroplateHandler (Caliper, Inc.). Plates are then incubated under establishedoptimal conditions of time and temperature. Then, a predeterminedquantity of the cell medium is transferred to a fresh microplate withthe Sciclone robot to generate an appropriate dilution. Luciferasesubstrate is added by means of a Wellmate reagent dispenser, andluminescence is measured in an Envision Multilabel microplate reader(PerkinElmer).

Example 11 Optimized Screening of Inhibitors

The system used to screen inhibitors is subjected to a pilot screen toassess screening conditions. The optimized assay configuration is testedin a pilot screen of ˜2,000 compounds at 2-3 different concentrations.Controls are included in each plate—8 wells for 0% inhibition (DMSOonly) and 8 wells for nearly complete inhibition (BoNT/A-LC inhibitor orsiRNA for BoNT/B-LC). Assay plates receive appropriate reporter cellsand compounds to be tested according to the protocol described above.The data obtained from this screen is used to determine variation (%CV), the hit rate at various z-score cutoffs, and may identify anyproblems with the assay which require resolution before HTS begins. Thedata from the pilot screen is then used to determine the compoundconcentration for the screen (probably in the range of 25-40 μM) inorder to establish a hit rate between 0.1% and 1%. The criteria fordesignating a compound as a hit is determined in the pilot screen;however, a z-score >3 or >5 is likely suitable. The z-score for eachsample is derived by subtracting the sample RLU from the mean negativecontrol RLU and dividing the difference by the negative control standarddeviation.

Example 12 Screening for Inhibitors

The method in accordance with one embodiment of the present inventionmay also be utilized to screen diverse compound libraries to identifyand confirm protease inhibitors with IC50's of ≦10 μM.

The high-throughput cellular BoNT/A-LC and BoNT/B-LC screens describedabove, is applied to libraries of discrete small molecules and naturalproducts in order to identify compounds having potent inhibitoryactivity against either of these botulinum neurotoxins. Hits from thescreen are confirmed by re-assay, establishing that they inhibit eitherBoNT/B-LC or BoNT/A-LC, but not both, and by demonstrating their potencyin concentration-dependent inhibition studies (IC50).

A. Compound libraries and sample handling.

The NERCE library. The compound collections of the National ScreeningLaboratory (NSRB) of the New England Regional Center for Excellence forBiodefense and Emerging Infectious Diseases (NERCE/BEID) at HarvardMedical is used as one example of a small molecule library to be screenin the cleave off cell based BoNT screening system. This library hasbeen assembled by a group of NERCE's chemistry consultants who screenedout compounds with undesirable properties, such as poor solubility,potential detergent-like activities, lack of stability in aqueoussolutions and chemical reactivity. There are currently ˜165,000compounds available including some that overlap with our in-housecollection. The overlap between the two libraries is ≦10%. Therefore,the combined library resource represent ˜300,000 distinct compounds.

B. Application of the primary BoNT/A&B-LC screens. Compounds in acandidate chemical library are examined in 96 or 384-well format vs. thecell-based BoNT/A-LC and BoNT/B-LC cell base HTS described above.Screening library compounds are stored in 96-well master plates at 2.5mM in 100% DMSO at −20° C. Master plates are thawed, and an amount ofcompound determined in the pilot screen described above are added to theassay plates by means of a SciClone ALH 3000 liquid handling robot(Caliper, Inc.) and a Twister II Microplate Handler (Caliper, Inc.), atthe same time, combining 4×96-well source plates into one 384-well assayplate. The screening plates contain positive and negative controls inthe first and last columns as described for the pilot screen above.

Raw data generated by the plate reader is processed as follows: relativeluminescence unit (RLU) data is captured and analyzed in asemi-automated procedure by relating the plate serial number to thedatabase entry, associating the numerical readout to each compoundentry, and calculating the % inhibition and z-score. In addition, aZ′-factor calculation is performed on each plate based on the positiveand negative controls; Z′ values of >0.6 are considered adequate, anddata from compounds in that plate are accepted into the database. Allscreening data, including the % inhibition, z-score, andconfirmation/validation data such as the 50% inhibitory concentration(IC50) and the counter-screen results is stored in one central database(CambridgeSoft's ChemBioOffice). A structure-activity relationship on aninvestigated chemical series is analyzed quickly. In addition, analogcompounds are identified rapidly from commercial databases, acquired,registered into the database and submitted for biological testing.

C. Hit confirmation and verification. Compounds that satisfy thecriteria for designation as primary hits undergo a 3-step confirmationprocess previously described. First, primary hits are selected fromstock plates into a confirmation stock plate and replicated to produce aset of 4 confirmation assay plates. The 4 confirmation assay plates areused in the primary screening assay to generate 4 new data points foreach compound. A confirmed hit displays inhibition >50% and a z-score >3in at least 3 of the 4 replicated assays. Second, confirmed hits arecounter-screened in replicate for inhibition of the other botulinumneurotoxin. Third, confirmed hits may be examined forconcentration-dependent activity in FRET assays for inhibition ofBoNT/A-LC and BoNT/B-LC; an IC50 is determined to rank the potency ofeach.

Due to the reliability of gain-of-signal cell-based assays, few falsepositives are encountered in the screens if the Z′ factor is >0.5throughout the screening process. False-negatives may arise due toinhibition of both the botulinum neurotoxin and processes required forgeneration of bioluminescence. However, these hits would likely bepromiscuous and not of sufficient quality to pursue even if they weredetectable. Hits that pass the counter-screen with the alternatebotulinum neurotoxin will likely not be promiscuous. To validate andprioritize the primary hits, several secondary assays are applied asdescribed below to further qualify the hits from the screen. If the hitrate is below 0.1% using the criteria established above, the hit ratemay be increased by accepting lower inhibition levels for hits as longas the Z′ value for the screening plate is above ˜0.6, indicating a wideseparation band between the negative and positive controls, and each hitis at least 3 standard deviations below the fully active control.

In the next step of a method in accordance with one embodiment of thepresent invention, each identified inhibitor is validated and multiplehits are prioritized by potency and selectivity. It is contemplated thatvalidated inhibitors of BoNT/A and BoNT/B, may have IC50s of ≦10 μM, aselectivity index CC50/IC50≧10, no significant cytotoxicity, anddemonstrated activity in primary neuronal cell model.

This step in one embodiment of the present invention generates thepotency and specificity information necessary to prioritize screeninghits/or chemotypes discovered in the HTS assays described above. In onepreferred embodiment four types of activity may be assessed: (a) invitro potency (IC50 for inhibition of the BoNT/A and BoNT/Bendopeptidase activities in vitro), (b) specificity (IC50 for potency ofinhibition of other endopeptidases in vitro, BoNT/F, anthrax lethalfactor (AT-LF), and a panel of human matrix metalloproteases, MMP's; andtest of chelation properties), (c) cytotoxicity (i.e., CC50 of thecompounds on mammalian cells in culture), and (d) in vivo potency (i.e.,IC50 for inhibition of the BoNT/A SNAP-25 cleavage or BoNT/B inhibitionof VAMP cleavage activity in primary rat neurons; and rescue of axonalgrowth inhibition). Successful compounds exhibit little or no detectablecytotoxicity or activity on other unrelated endopeptidases, but providepotent and specific rescue of BoNT/A and/or BoNT/B action in vitro andin isolated neurons.

Rat Neuronal Cell SNAP-25 Cleavage Assay. As described previously, cellsare harvested from 7-8 day old rat cerebella, washed and cultured in6-well plates, and grown over a week with media changes. Once the cellshave become networked neutrally, they are preincubated with compounds ordiluent (DMSO) for 15 min. Cells are then inoculated with BoNT/A andincubated for 3 hours at 37° C., 5% CO₂ before harvesting. Cells aretreated with 1 M NaOH to inactivate the BoNT and are scraped from theplate surface prior to centrifugation and lysis with a gel loadingbuffer. Samples are run on SDS-PAGE gels and then transferred tomembranes for immunoblot analysis with rabbit anti-SNAP-25 and thenHRP-conjugated goat anti-rabbit IgG. Band intensities are read andnormalized using scanning densitometry.

The invention has been described with references to preferredembodiments. While specific values, relationships, materials and stepshave been set forth for purposes of describing concepts of theinvention, it will be appreciated by persons skilled in the art thatnumerous variations and/or modifications may be made to the invention asshown in the specific embodiments without departing from the spirit orscope of the basic concepts and operating principles of the invention asbroadly described. It should be recognized that, in the light of theabove teachings, those skilled in the art can modify those specificswithout departing from the invention taught herein. Having now fully setforth the preferred embodiments and certain modifications of the conceptunderlying the present invention, various other embodiments as well ascertain variations and modifications of the embodiments herein shown anddescribed will obviously occur to those skilled in the art upon becomingfamiliar with such underlying concept. It is intended to include allsuch modifications, alternatives and other embodiments insofar as theycome within the scope of the appended claims or equivalents thereof. Itshould be understood, therefore, that the invention may be practicedotherwise than as specifically set forth herein. Consequently, thepresent embodiments are to be considered in all respects as illustrativeand not restrictive.

1. A system for the identification of proteases and protease inhibitors,comprising: a reporter construct comprising at least one binding site,an inducible promoter region, and at least one reporter gene, allfunctionally connected for expression of the reporter gene(s), infunctional coordination with a transcriptional activation agent; thetranscriptional activation agent comprising a nucleic acid bindingdomain, at least one protease substrate domain, and at least onetranscriptional activation domain for an inducible promoter.
 2. Thesystem of claim 1, further comprising at least one protease or proteasecandidate, wherein the protease or protease candidate specificallytargets the protease substrate domain.
 3. The system of claim 2, whereinat least one of the transcriptional activation agent or theprotease/protease candidate are provided into the system as products ofnucleic acid constructs.
 4. The system of claim 2, wherein at least oneof the transcriptional activation agent or the protease/proteasecandidate are provided into the system as a protein.
 5. The system ofclaim 1, further comprising at least one reporter gene selected from thegroup consisting of a Venus yellow fluorescent protein, yellowfluorescent protein (YFP), green fluorescent protein (GFP), cyanfluorescent protein (CFP); blue fluorescent protein (BFP), redfluorescent protein (RFP), fluorescing mutants thereof, bioluminescentproteins, Gaussia luciferase, renilla luciferase, click beetleluciferase, and firefly luciferase may also be used to quantify theactivity of the reporter vector.
 6. The system of claim 5 comprising atleast two reporter genes transcribed from one inducible promoter.
 7. Thesystem of claim 1, wherein the reporter gene construct comprises one toeight binding site sequence repeats.
 8. The system of claim 1, whereinthe reporter gene construct comprises five binding site sequencerepeats.
 9. The system of claim 1, wherein the binding site is selectedfrom the group consisting of Gal4 and LexA.
 10. The system of claim 1,wherein the inducible reporter region comprises a TATA promoter region.11. The system of claim 1, wherein the protease substrate is selectedfrom the group consisting of BoNT protease substrate, anthrax protease,caspase, alpha virus NSP2 protease, HIV processing proteases, SUMOprocessing proteases, ubiquitin processing proteases, ISG15 processingproteases, autophagy related ATG4 like processing proteases andHepatitis virus processing proteases or a combination thereof.
 12. Thesystem of claim 10, wherein the protease substrate is selected from thegroup consisting of SNAP-25, VAMP-2, and Syntaxin1a or a combinationthereof.
 13. The system of claim 10, wherein the protease substratecomprises SNAP-25 and VAMP-2 protease substrate domains.
 14. The systemof claim 11 wherein the protease is a BoNT protease.
 15. The system ofclaim 1, wherein the protease substrate is located on thetranscriptional activator agent between the binding domain and thetranscriptional activator domain and the transcriptional activationagent is in a cellular compartment where transcription takes place. 16.The system of claim 1, wherein the protease substrate domain is at oneend of the molecule and the binding domain and the transcriptionalactivator domain are located in functional proximity to each other andat an opposite end of the transcriptional activator agent from theprotease substrate domain, such as cleavage of the protease substratedomain releases the binding domain and the transcriptional activatorrendering a functional transcriptional activator agent fragment.
 17. Thesystem of claim 15, engineered such as the transcriptional activatoragent is sequestered outside of a cell nucleus but cleavage with aprotease releases a functionally active transcriptional activator agentfragment, comprising the binding domain and the transcriptionalactivator domain and capable to reach a cellular compartment wheretranscriptional activity takes place.
 18. The system of claim 2 in a tTScell.
 19. A method to identify protease inhibitors, comprising:introducing at least one test molecule or compound to a systemcomprising: a reporter gene construct comprising at least one bindingsite, a transcriptional promoter, an inducible promoter region, and atleast one reporter gene, all functionally connected for expression ofthe reporter gene(s) in functional coordination with a transcriptionalactivation agent, the transcriptional activation agent comprising anucleic acid binding domain, at least one protease substrate domain, andat least one transcriptional activation domain for an induciblepromoter, and a protease specific for the protease substrate domain; andmeasuring the effect on the level of expression of the reportermolecule.
 20. A method to identify a protease, comprising: introducingat least one test molecule or compound to a system comprising: areporter gene construct comprising at least one binding site, atranscriptional promoter, an inducible promoter region, and at least onereporter gene, all functionally connected for expression of the reportergene(s) in functional coordination with a transcriptional activationagent, the transcriptional activation agent comprising a nucleic acidbinding domain, at least one protease substrate domain, and at least onetranscriptional activation domain for an inducible promoter, andmeasuring the effect on the level of expression of the reportermolecule.